Tyrosine phosphorylation is a key mechanism for transmitting growth signals into cells. In T lymphocytes, both the T cell antigen receptor (TCR) and the receptor for the growth factor interleukin-2 (IL-2) use multiple well known protein tyrosine kinases (PTKs) for signaling. In contrast, very little is known about the protein tyrosine phosphatases (PTPases) that counteract these PTKs by dephosphorylating them and their substrates. This proposal focuses on the PEP PTPase and the Csk PTK, which form a complex that negatively regulates early TCR signaling events. 1) First, we will generate and analyze PEP-/-, antigen-specific, CD4+ and CD8+ T cells by crossing PEP knock-out mice with TCR transgenic animals. We will also generate PEP-/- clones of the Jurkat T leukemia cell line by a novel somatic gene deletion technology. We will determine to what extent TCR signaling is altered in the absence of PEP and if Lck and Fyn are hyperactive. As a control, we will use the IL-2R system to test the notion that PTKs that do not depend on Src family kinases, such as Jakl and Jak3, are insensitive to loss of PEP. 2) Second, we will use the PEP-l- T cells to gain some insight into the structure-function relationships, specificity, and subcellular targeting of the PEP-Csk complex to lipid rafts or two interacting proteins, PAG/Cbp and G3BP. 3) Third, we will continue to study our finding that PKA phosphorylates Csk at serine 364 ($364) in vitro as well as in intact T cells and thereby activates Csk 3- to 5-fold. This activation results in increased suppression of Lck and reduced TCR signaling. We will investigate whether PEP is also directly phosphorylated at $35 and regulated by PKA. The molecular mechanism by which PEP is regulated will also be examined. 4) Fourth, we will undertake a number of structural studies and will attempt to solve the crystal structure of individual domains and their complexes, ultimately hoping to understand the structure of the whole enzymes and even their complex. These studies are particularly important ifor understanding the regulation of PEP and the impact of PKA-mediated phosphorylation of Csk at $364. Overall, these studies will provide a detailed characterization of the function and structure of PEP and Csk from the level of physiological relevance in whole animals to atomic structure and regulation. These issues are important for our understanding of the molecular machinery of T cell activation and the immune response.